Department of Cell Biology and Development, Biomedical Sciences Institute, University of Sao Paulo, Sao Paulo, Brazil.
Invest Ophthalmol Vis Sci. 2021 Mar 1;62(3):31. doi: 10.1167/iovs.62.3.31.
Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues.
Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2.
Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2.
This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.
成年哺乳动物眼睛的睫状上皮（CE）含有静止的视网膜祖细胞/干细胞，这些细胞在体外产生神经球，并分化为视网膜神经元。这种能力不能有效地进化，可能是由于调节机制，如 microRNAs（miRNAs），它们控制多能、祖细胞和分化基因。在这里，我们研究了 Let-7 miRNAs 及其调节剂和靶标 Lin28 和 Hmga2 在神经球、新生和成年组织中的 CE 细胞中的存在。
将新生和成年大鼠的 CE 细胞从色素上皮（PE）和非色素上皮（NPE）中分离出来。新生的 PE 细胞在生长因子的作用下培养形成神经球，我们分析了 Let-7、Lin28a 和 Hmga2 的表达。在神经球形成过程中，我们添加了化学修饰的单链寡核苷酸，旨在结合并抑制或模拟内源性成熟的 Let-7b 和 Let-7c。在培养 7 天后，我们分析了神经球的大小、数量以及 Let-7、Lin28 和 Hmga2 的表达。
Let-7 miRNAs 在新生 CE 细胞中的表达水平较低，在成年组织中的表达水平显著增加，在 NPE 细胞中的表达水平更高，但不具有干细胞重编程能力。Lin28a 和 Hmga2 蛋白和转录本在新生细胞中的表达水平高于成年细胞，而 Let-7 则相反。神经球的 Lin28 和 Hmga2 表达水平高于新生和成年细胞，但与新生细胞相比，Let-7 的表达水平相似。Let-7b 抑制剂上调了 Hmga2 的表达，而 Let-7c 模拟物上调了 Lin28 的表达，下调了 Hmga2 的表达。
本研究显示了 Lin28-Let-7-Hmga 调节轴在 CE 细胞中的动态变化。这些成分在神经球形成和出生后 CE 细胞中可能发挥不同的作用。
